PDE4 associates with different scaffolding proteins: modulating interactions as treatment for certain diseases.

cAMP is an ubiquitous second messenger that is crucial to many cellular processes. The sole means of terminating the cAMP signal is degradation by cAMP phosphodiesterases (PDEs). The PDE4 family is of particular interest because PDE4 inhibitors have therapeutic potential for the treatment of various inflammatory and auto-immune diseases and also have anti-depressant and memory-enhancing effects. The subcellular targeting of PDE4 isoforms is fundamental to the compartmentalization of cAMP signaling pathways and is largely achieved via proteinprotein interactions. Increased knowledge of these protein-protein interactions and their regulatory properties could aid in the design of novel isoform-specific inhibitors with improved efficacy and fewer prohibitive side effects.

cAMP phosphodiesterase-4A1 (PDE4A1) has provided the paradigm for the intracellular targeting of phosphodiesterases, a process that underpins compartmentalized cAMP signalling.

Specificity of cAMP signalling pathways has shown that the intracellular targeting of the individual components confers a three-dimensional context to the signalling paradigms in which they can exquisitely control the specificity of the outcome of the signal. Pivotal to this paradigm is degradation of cAMP by sequestered PDEs (phosphodiesterases). cAMP rapidly diffuses within cells and, without the action of spatially confined PDE populations, cAMP gradients could not be formed and shaped within cells so as to regulate targeted effector proteins. Of particular importance in regulating compartmentalized cAMP signalling are isoforms of the PDE4 family, which are individually defined by unique N-terminal regions. We have developed and pioneered the concept that a major function of this N-terminal region is to confer intracellular targeting of particular PDE4 isoforms on specific signalling complexes and intracellular locations. The paradigm for this concept developed from our original studies on the PDE4A1 (RD1) isoform. The N-terminal region unique to PDE4A1 consists of two well-defined helical regions separated by a mobile hinge region. Helix-2 provides the core membrane-insertion module, with helix-1 facilitating membrane association and fidelity of targeting in living cells. The irreversible, Ca(2+)-dependent insertion of the N-terminal region of PDE4A1 into membranes provides 'long-term' memory of cell activation.

Molecular cloning, genomic positioning, promoter identification, and characterization of the novel cyclic amp-specific phosphodiesterase PDE4A10.

We describe the cloning and expression of HSPDE4A10, a novel long form splice variant of the human cAMP phosphodiesterase PDE4A gene. The 825 amino acid HSPDE4A10 contains a unique N terminus of 46 amino acids encoded by a unique 5' exon. Exon-1(4A10) lies approximately 11 kilobase pairs (kb) downstream of exon-1(4A4) and approximately 13.5 kb upstream of the PDE4A common exon 2. We identify a rat PDE4A10 ortholog and reveal a murine ortholog by nucleotide sequence database searching. PDE4A10 transcripts were detected in various human cell lines and tissues. The 5' sequence flanking exon-1(4A10) exhibited promoter activity with the minimal functional promoter region being highly conserved in the corresponding mouse genomic sequence. Transient expression of the engineered human PDE4A10 open reading frame in COS7 cells allowed detection of a 121-kDa protein in both soluble and particulate fractions. PDE4A10 was localized primarily to the perinuclear region of COS7 cells. Soluble and particulate forms exhibited similar K(m) values for cAMP hydrolysis (3-4 microM) and IC(50) values for inhibition by rolipram (50 nM) but the V(max) value of the soluble form was approximately 3-fold greater than that of the particulate form. At 55 degrees C, soluble HSPDE4A10 was more thermostable (T(0.5) = 11 min) than the particulate enzyme (T(0.5) = 5 min). HSPDE4A10 and HSPDE4A4B are shown here to be similar in size and exhibit similar maximal activities but differ with respect to sensitivity to inhibition by rolipram, thermostability, interaction with the SRC homology 3 domain of LYN, an SRC family tyrosyl kinase, and subcellular localization. We suggest that the unique N-terminal regions of PDE4A isoforms confer distinct properties upon them.

The cAMP-specific phosphodiesterase PDE4A5 is cleaved downstream of its SH3 interaction domain by caspase-3. Consequences for altered intracellular distribution.

The unique N-terminal region of the cAMP-specific phosphodiesterase PDE4A5, which confers an ability to bind to certain protein SH3 domains, is cleaved during apoptosis in both Rat-1 fibroblasts and PC12 cells. Cleavage was abolished by the caspase-3-selective inhibitor, z-DEVD-CHO but not the caspase-1 selective inhibitor, z-YVAD-CHO. Caspase-3 treatment of PDE4A5, expressed either transiently in COS cells or generated in vitro by coupled transcription translation, generated a similar cleavage product of 100 kDa compared with the native 110-kDa PDE4A5. This product could be detected immunochemically with an antibody raised to a C-terminal PDE4A5 peptide but not an antibody raised to the N terminus of PDE4A5, indicating that caspase-3 caused N-terminal cleavage of PDE4A5. Deletion of the putative caspase-3 cleavage site, (69)DAVD(72), in PDE4A5, or generation of either the D72A or the D69A mutants, ablated the ability of caspase-3 to cause cleavage. The N-terminal truncate PDE4A5-DeltaP3 was engineered to mimic the caspase-cleaved product of PDE4A5. This showed altered catalytic activity and, unlike PDE4A5, was unable to interact with the SH3 domain of the tyrosyl kinase, LYN. Although both PDE4A5 and PDE4A5-DeltaP3 were localized at cell cortical regions (ruffles), the distinct perinuclear association noted for both PDE4A5 and LYN was not seen for PDE4A5-DeltaP3. Staurosporine-induced apoptosis caused a marked redistribution of PDE4A5 but not PDE4A8 in stably transfected Rat-1 cells. The PDE4-selective inhibitor, rolipram together with the adenylyl cyclase activator forskolin, caused a synergistic increase in the apoptosis of Rat-1 cells. Overexpression of PDE4A5 in Rat-1 cells protected against staurosporine-induced apoptosis in contrast to overexpression of PDE4A8, which potentiated apoptosis. PDE4A5 may be the sole PDE4 family member to provide a substrate for caspase-3 cleavage and this action serves to remove the SH3 binding domain that is unique to this isoform within the PDE4A family and to alter its intracellular targeting.

Association with the SRC family tyrosyl kinase LYN triggers a conformational change in the catalytic region of human cAMP-specific phosphodiesterase HSPDE4A4B. Consequences for rolipram inhibition.

The cAMP-specific phosphodiesterase (PDE) HSPDE 4A4B(pde46) selectively bound SH3 domains of SRC family tyrosyl kinases. Such an interaction profoundly changed the inhibition of PDE4 activity caused by the PDE4-selective inhibitor rolipram and mimicked the enhanced rolipram inhibition seen for particulate, compared with cytosolic pde46 expressed in COS7 cells. Particulate pde46 co-localized with LYN kinase in COS7 cells. The unique N-terminal and LR2 regions of pde46 contained the sites for SH3 binding. Altered rolipram inhibition was triggered by SH3 domain interaction with the LR2 region. Purified LYN SH3 and human PDE4A LR2 could be co-immunoprecipitated, indicating a direct interaction. Protein kinase A-phosphorylated pde46 remained able to bind LYN SH3. pde46 was found to be associated with SRC kinase in the cytosol of COS1 cells, leading to aberrant kinetics of rolipram inhibition. It is suggested that pde46 may be associated with SRC family tyrosyl kinases in intact cells and that the ensuing SH3 domain interaction with the LR2 region of pde46 alters the conformation of the PDE catalytic unit, as detected by altered rolipram inhibition. Interaction between pde46 and SRC family tyrosyl kinases highlights a potentially novel regulatory system and point of signaling system cross-talk.

Counteracting HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with XV638 and SD146, cyclic urea amides with broad specificities.

The long-term therapeutic benefit of HIV antiretroviral therapy is still threatened by drug-resistant variants. Mutations in the S1 subsite of the protease are the primary cause for the loss of sensitivity toward many HIV protease inhibitors, including our first-generation cyclic urea-based inhibitors DMP323 and DMP450. We now report the structures of the three active-site mutant proteases V82F, I84V, and V82F/I84V in complex with XV638 and SD146, two P2 analogues of DMP323 that are 8-fold more potent against the wild type and are able to inhibit a broad panel of drug-resistant variants [Jadhav, P. K., et al. (1997) J. Med. Chem. 40, 181-191]. The increased efficacy of XV638 and SD146 is due primarily to an increase in P2-S2 interactions: 30-40% more van der Waals contacts and two to four additional hydrogen bonds. Furthermore, because these new interactions do not perturb other subsites in the protease, it appears that the large complementary surface areas of their P2 substituents compensate for the loss of P1-S1 interactions and reduce the probability of selecting for drug-resistant variants.

Molecular recognition of cyclic urea HIV-1 protease inhibitors.

As long as the threat of human immunodeficiency virus (HIV) protease drug resistance still exists, there will be a need for more potent antiretroviral agents. We have therefore determined the crystal structures of HIV-1 protease in complex with six cyclic urea inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an attempt to identify 1) the key interactions responsible for their high potency and 2) new interactions that might improve their therapeutic benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored in the active site of the protease by six hydrogen bonds and that their P1 and P2 substituents participate in extensive van der Waals interactions and hydrogen bonds. Because all of our inhibitors possess benzyl groups at P1 and P1', their relative binding affinities are modulated by the extent of their P2 interactions, e.g. XK216, the least potent inhibitor (Ki (inhibition constant) = 4.70 nM), possesses the smallest P2 and the lowest number of P2-S2 interactions; whereas SD146, the most potent inhibitor (Ki = 0.02 nM), contains a benzimidazolylbenzamide at P2 and participates in fourteen hydrogen bonds and approximately 200 van der Waals interactions. This analysis identifies the strongest interactions between the protease and the inhibitors, suggests ways to improve potency by building into the S2 subsite, and reveals how conformational changes and unique features of the viral protease increase the binding affinity of HIV protease inhibitors.

Intracellular compartmentalization of PDE4 cyclic AMP-specific phosphodiesterases.

The PDE4 cyclic AMP-specific phosphodiesterase family comprises a large number of different isoforms encoded by four distinct genes, with additional complexity arising through alternate mRNA splicing. This generates a number of distinct PDE4 isoforms with unique N-terminal regions. The range of such splice variants emanating from the four PDE4 genes appears to be highly conserved across species. One key role for such regions appears to be their potential to target isoforms to specific intracellular sites. Evidence for such a targeting role for these N-terminal regions can be gleaned by a variety of techniques. These include subcellular fractionation, confocal microscopy, binding assays to show association with proteins having src homology 3 (SH3) domains, and generation of chimeric constructs of these N-terminal regions with proteins that are normally expressed in the cytosol.

Molecular basis of HIV-1 protease drug resistance: structural analysis of mutant proteases complexed with cyclic urea inhibitors.

In cell cultures, the key residues associated with HIV-1 resistance to cyclic urea-based HIV-1 protease (PR) inhibitors are Val82 and Ile84 of HIV-1 PR. To gain an understanding of how these two residues modulate inhibitor binding, we have measured the Ki values of three recombinant mutant proteases, I84V, V82F, and V82F/I84V, for DMP323 and DMP450, and determined the three-dimensional structures of their complexes to 2.1-1.9 A resolution with R factors of 18.7-19.6%. The Ki values of these mutants increased by 25-, 0.5-, and 1000-fold compared to the wild-type values of 0.8 and 0.4 nM for DMP323 and DMP450, respectively. The wild-type and mutant complexes overall are very similar (rms deviations of 0.2-0.3 A) except for differences in the patterns of their van der Waals (vdw) interactions, which appear to modulate the Ki values of the mutants. The loss of the CD1 atom of Ile84, in the I84V mutant complexes, creates a hole in the S1 subsite, reducing the number of vdw contacts and increasing the Ki values. The V82F mutant binds DMP323 more tightly than wild type because the side chain of Phe82 forms additional vdw and edge-to-face interactions with the P1 group of DMP323. The Ki values of the single mutants are not additive because the side chain of Phe82 rotates out of the S1 subsite in the double mutant (the chi 1 angles of Phe82 and -182 in the V82F and V82F/I84V mutants differ by 90 and 185 degrees, respectively), further reducing the vdw interactions. Finally, compensatory shifts in the I84V and V82F/ I84V complexes pick up a small number of new contacts, but too few to offset the initial loss of interactions caused by the mutations. Therefore, our data suggest that variants persist in the presence of DMP323 and DMP450 because of a decrease in vdw interactions between the mutant proteases and inhibitors.

Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3.

5'-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5' sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue 'long-form' PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 microM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 microM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 microM) to rolipram inhibition than its cytosolic form (IC50 0.2 microM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.

Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditioned rat heart.

The stable prostacyclin derivative, 7-oxo-prostacyclin, exhibits a delayed, long-lasting cardioprotective effect, which is accompanied by an increase in cyclic nucleotide phosphodiesterase (PDE) activities restricted to the Ca2+-calmodulin-dependent (PDE1) and cyclic AMP-specific phosphodiesterase (PDE4) activities. Mammalian PDEs form a large multi-gene family with differential expression occurring in a cell- and tissue-specific manner. The aim of this study was to identify which isoforms of PDE1 and PDE4 are present in the hearts of control and 7-oxo-prostacyclin treated rats. Using RT-PCR analysis, we were able to identify in control rat hearts transcripts for PDE1C, but not for either PDE1A or PDE1B within the three-gene PDE1 family. Within the four-gene PDE4 family we detected, by generic RT-PCR analysis, transcripts for PDE4A, PDE4B and PDE4D, but not PDE4C. Using RT-PCR primers for specific splice variants, we identified transcripts for PDE4B1, PDE4B2, PDE4B3, PDE4D1, PDE4D2 and PDE4D3 in hearts from the control animals. Immunoblotting of hearts from the control animals for PDE4 forms allowed us to identify a 98-kDa PDE4A species, 68-kDa band representing PDE4D1/2 and a 95-kDa species indicative of PDE4D3. In the hearts of treated animals, 48 h after a single 50 microgram/kg dose of 7-oxo-prostacyclin, a profound increase in transcript levels was seen for both PDE1C and PDE4B3 together with a slight elevation for PDE4B1. No change in PDE4A transcripts occurred, which was consistent with a lack of change indicated in immunoblotting analyses. In contrast, 7-oxo-prostacyclin treatment caused decrease in transcript levels for PDE4D, which was confirmed by immunoblotting and shown to be due to a reduction in the levels of PDE4D3 and also in PDE4D1/D2. Thus, treatment of animals with 7-oxo-prostacyclin initiated profound isoform-specific changes in PDE expression in the myocardium which, presumably, underpin the increased PDE activity.

The human cyclic AMP-specific phosphodiesterase PDE-46 (HSPDE4A4B) expressed in transfected COS7 cells occurs as both particulate and cytosolic species that exhibit distinct kinetics of inhibition by the antidepressant rolipram.

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed approximately 96% of the total COS cell PDE activity. A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells. Immunoblotting studies identified PDE-46 as a approximately 125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was approximately 56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations. Immunofluorescence analysis by laser scanning confocal microscopy showed that PDE-46 was located at discrete margins of the cell, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells. h6.1 was shown to have an approximately 11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0. 195 microM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 microM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 microM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 microM; Ki' = 2.3 microM). Particulate PDE-46 thus showed a approximately 60-fold higher affinity for rolipram than cytosolic PDE-46.

Production, purification, and crystallization of human interleukin-1 beta converting enzyme derived from an Escherichia coli expression system.

Interleukin-1 beta converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL-1 beta to an active mature form. The mature form of IL-1 beta is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273-277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter-based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X-ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.

The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation.

In this study, we have examined both the ability of various Ca2+ channel sub-types to support the release of [3H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (-)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [3H]glutamate by K(+)-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 microM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, omega-conotoxin GVIA; however, the P/Q-type calcium channel blocker omega-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with omega-conotoxin GVIA. K(+)-stimulated release of glutamate was observed when stimulated either in the presence of Ca2+ or of Ba2+ and similar inhibition of release by (-)-baclofen was seen under both conditions. In contrast to these results, ionomycin-evoked glutamate release was greatly reduced as compared to K(+)-evoked release and was not modulated by (-)-baclofen. In the presence of omega-conotoxin GVIA alone, inhibition of release by (-)-baclofen was attenuated but not abolished. Following block of nicardipine-sensitive channels, inhibition of release by (-)-baclofen was still present, and after prior block of omega-conotoxin GVIA-sensitive channels the presence of nicardipine restored the ability of (-)-baclofen to inhibit residual release of glutamate. Modulation of glutamate release by (-)-baclofen was unaffected by the presence of omega-agatoxin IVA alone; however, after block of both omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive channels, inhibition of release by (-)-baclofen was completely abolished. These results indicate that multiple sub-types of voltage-dependent calcium channels are present on the presynaptic terminals of cerebellar granule neurons and support K(+)-stimulated release of [3H]glutamate. Modulation of release by GABAB receptor activation appears to be dependent upon interaction of this receptor with a number of voltage-sensitive calcium channels, including omega-conotoxin GVIA-sensitive and omega-agatoxin IVA-sensitive channels.

Production of active human interleukin-1 beta-converting enzyme in a baculovirus expression system.

The cDNA coding for the precursor form of human interleukin-1 beta-converting enzyme (proICE) was expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The 45-kDa recombinant protein was further processed to several smaller forms of 32, 24, 20, 13 and 10 kDa. Active recombinant ICE derived from the baculovirus expression system (bvICE) was found to be present in soluble lysates of insect cells as an associated heterodimer consisting of 10- and 20-kDa subunits. The activity of bvICE was determined by conversion of precursor interleukin-1 beta (preIL-1 beta) to the mature form (mIL-1 beta) and via site-specific cleavage of a decapeptide which spans the ICE cleavage site in preIL-1 beta. The bvICE system was inhibited by an ICE inhibitor to the same extent as native ICE from the monocytic cell line THP-1. Expression of an active-site mutant (Cys285 to Ser) of proICE in insect cells resulted in the accumulation of partially processed (32-kDa) ICE. The availability of a facile expression system will permit further characterization of the biochemical properties and processing pathway of this unique protease.

Photochemical labeling and inhibition of Na,K-ATPase by 2-Azido-ATP. Identification of an amino acid located within the ATP binding site.

2-Azido-ATP (2-N3-ATP) was investigated as a reagent for the identification of amino acids located within the catalytic ATP binding site of Na,K-ATPase. The enzymatic activity of Na,K-ATPase was inhibited up to 50% by 2-N3-ATP (K0.5 = 5-10 microM) after irradiation with ultra-violet light, and inhibition was prevented by 0.2 mM ATP. The binding of ATP to Na,K-ATPase (KD = 0.1 microM) was inhibited competitively by 2-N3-ATP. [alpha-32P]2-N3-ATP labels the alpha subunit of Na,K-ATPase, and the stoichiometry of covalent ATP-protectable incorporation of the probe into the protein is approximately equal to the stoichiometry of high-affinity binding of ATP to the Na,K-ATPase. 2-N3-ATP is also hydrolyzed by Na,K-ATPase as a substrate. From these data, it is concluded that 2-N3-ATP photochemically labels the Na,K-ATPase from within the catalytic ATP site on the protein. Trypsin digestion of Na,K-ATPase after photochemical labeling with [alpha-32P]2-N3-ATP generated a large 30-kDa fragment containing the radiolabeled nucleotide. This fragment was resistant to further cleavage by trypsin, but it could be digested further after denaturation in urea. High pressure liquid chromatography separation of tryptic peptides from the 30-kDa fragment and subsequent amino acid sequence analysis of the radiolabeled peptides identified the region between His496 and Arg510 of the Na,K-ATPase alpha subunit as the region labeled by [alpha-32P]2-N3-ATP. Gly502 was absent from all sequences of the radiolabeled peptides from this region, consistent with the derivatization of this amino acid by 2-N3-ATP and localization of Gly502 within the ATP binding site.

Cycloheximide abolishes pertussis toxin-induced increase in glutamate release from cerebellar granule neurones.

Release of glutamate from cerebellar granule neurones was stimulated either by adding 50 mM K+ to normal Krebs medium, or by adding 5 mM Ca2+ to neurones continuously depolarised with 50 mM K+ in the absence of Ca2+. Pre-incubation of neurones for 16 h with pertussis toxin (PTX) increased the stimulated glutamate release in both K(+)-stimulated and continuously depolarised neurones. Under both conditions, the PTX-induced increase in release was abolished by cycloheximide. In contrast, in the presence of cycloheximide, PTX still prevented the GABAB agonist (-)-baclofen from inhibiting glutamate release. These results suggest that G-protein ADP-ribosylation by PTX in cerebellar granule neurones may increase synthesis of a protein associated with the L-type calcium channel.